Purification and Characterization of Human Kallikrein 11, a Candidate Prostate and Ovarian Cancer Biomarker, from Seminal Plasma

Abstract

Purpose: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11from seminal plasma. Experimental Design: hK11was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis,Western blots, and mass spectrometry.Results: hK11is present in seminal plasma at concentrations ranging from 2 to 37 Ag/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of f40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg 156 (Genbank accession no. AF164623), which generates two peptides of f20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, a 1 -antichymotrypsin, a 2 -antiplasmin, and a 1 -antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11activity up to 40%. Plasmin is a strong candidate for cleaving hK11at Arg .Conclusion: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

Activation and enzymatic characterization of recombinant human kallikrein 8

“…Primary substrate specificity analysis has been reported for the new members of the human tissue kallikrein family, hK4, 5, 6, 7, 11, 13, and 14 (Egelrud, 1993;Takayama et al, 2001;Magklara et al, 2003;Kapadia et al, 2004;Felber et al, 2005;Michael et al, 2005;Luo et al, 2006). All of them demonstrated trypsin-like activity, with the exception of the chymotrypsin-like protease hK7.…”

Kallikrein-related peptidases

“…3). A tryptic or Argrestricted specificity has also been confirmed with KLK2 [19,20], KLK4 [21][22][23], KLK5 [22][23][24][25][26], KLK6 [22,[27][28][29], KLK8 [30,31], KLK10 [22], KLK11 [22,32], KLK12 [33], KLK13 [23] and KLK14 [23,34,35]. The architecture of the catalytic cleft in KLK10, KLK11 and KLK14 also allow the enzymes to accept amino acid residues with large hydrophobic side chains, e.g., Tyr, Met, and Leu, similar to chymotrypsin.…”

Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers

“…Sandwich type KLK-specifi c ELISAs, with one monoclonal or polyclonal antibody used for capture and another polyclonal one used for detection have been developed for quantifi cation and detection of each KLK in tissue extracts and biological fl uid samples acquired for routine biochemical testing (9) . For instance, protein concentrations of KLK6 and KLK10 in ovarian cancer ascites fl uids were measured with ELISA-type immunoassays (144) . A very recent study established, also, a reference interval for KLK6 serum levels in adults (145) .…”